A compound microbial agent for improving the disease resistance of honeysuckle and a preparation method thereof
The use of compound microbial agents has solved the problem of pests and diseases caused by soil microbial imbalance in honeysuckle cultivation, enhanced the disease resistance of honeysuckle, and achieved effective control of soil-borne diseases.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- LINYI UNIVERSITY
- Filing Date
- 2026-03-20
- Publication Date
- 2026-07-14
AI Technical Summary
Imbalance in the soil microbial community during honeysuckle cultivation exacerbates pests and diseases. The use of chemical pesticides and fertilizers leads to an imbalance in the soil ecosystem, resulting in poor pest and disease control.
A compound microbial agent containing bacterial solutions of Bacillus pumilus, Priestella megaterium, and Bacillus rotundifolia, combined with resistant dextrin, glycerol, trehalose, and other ingredients, is used to enhance the disease resistance of honeysuckle through root irrigation or foliar spraying.
It significantly improves the disease resistance of honeysuckle, inhibits soil-borne pathogens, enhances defense capabilities, restores soil ecology, reduces disease damage, and achieves lasting control effects.
Smart Images
Figure SMS_1 
Figure SMS_2 
Figure SMS_3
Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial inoculant technology, and relates to a compound microbial inoculant for improving the disease resistance of honeysuckle and its preparation method. Background Technology
[0002] Honeysuckle (scientific name: *Lonicera japonica* Thunb.), also known as Lonicera japonica or Lonicera vine, is a perennial semi-evergreen vine belonging to the genus *Lonicera* in the family Caprifoliaceae. Honeysuckle is sweet and cold in nature, and functions to clear heat and detoxify, reduce inflammation and swelling. It is effective against bacterial dysentery and various suppurative diseases. "Honeysuckle dew" is an aromatic volatile oil and water-soluble extract from honeysuckle using distillation, and is an excellent remedy for clearing heat and detoxifying, treating infantile eczema, boils, fever, and thirst. Its stems and vines are also used medicinally.
[0003] Honeysuckle, a traditional Chinese medicine, plays an important role in traditional medicine due to its heat-clearing, detoxifying, anti-inflammatory, and antibacterial properties. With increasing market demand, the scale of honeysuckle cultivation is constantly expanding. However, this has led to an increase in pests and diseases, posing a serious threat to the yield and quality of honeysuckle. Common diseases include brown spot, powdery mildew, and root rot, all of which affect honeysuckle growth to varying degrees.
[0004] In intensive honeysuckle cultivation, the soil microbial community exhibits a significant structural imbalance. The abundance of beneficial bacteria such as actinomycetes and Bacillus has decreased, while the number of pathogens such as Fusarium and Rhizoctonia has increased. This imbalance directly leads to abnormal soil enzyme activity, creating a micro-ecological environment conducive to soil-borne diseases, resulting in increased incidence of root rot and white mold. Continuous monitoring shows that soil pH is 0.5–1.2 lower than in natural forests, and organic matter content has sharply decreased to below 12.8 g / kg, significantly weakening the diversity of microbial metabolism. The root cause of this soil microbial imbalance lies in the long-term and excessive use of chemical pesticides and fertilizers. Excessive nitrogen fertilizer application has caused nitrate nitrogen residues to exceed the 82 mg / kg threshold, directly inhibiting the activity of nitrogen-fixing bacteria. Broad-spectrum fungicides are used an average of 6–8 times per year, leading to a 19.7% decrease in the microbial genetic diversity index. These chemicals not only directly threaten the survival of beneficial microorganisms in the soil and inhibit their activity, but also damage the stability and function of the soil microbial community, causing an imbalance in the soil ecosystem.
[0005] Therefore, traditional control methods rely on chemical pesticides, but long-term use may lead to pesticide residues, environmental pollution, imbalance of soil ecosystems, and increased pesticide resistance in pests and diseases, which in turn affects the ecological balance. Seeking environmentally friendly and sustainable control measures has become crucial. Summary of the Invention
[0006] To address the above problems, this invention provides a compound microbial agent for improving the disease resistance of honeysuckle and its preparation method. The microbial agent can significantly improve the disease resistance of honeysuckle.
[0007] The present invention employs the following technical solutions to achieve the above objectives: A compound microbial agent for improving the disease resistance of honeysuckle, based on 1L of compound microbial agent, mainly contains the following components: 50-60mL of Bacillus puerariae bacterial solution, 80-100mL of Priestella megaterium bacterial solution, 50-60mL of Bacillus humifusa bacterial solution, 10-15g of resistant dextrin, 80-100g of glycerol, 20-30g of trehalose, 8-10g of sodium ascorbate, 20-30g of Tremella fuciformis oligopeptide, 40-50g of Phellinus linteus polysaccharide, 10-15mL of sugar alcohol silicon, 15-20mL of sugar alcohol zinc, and 20-30g of sodium polyglutamate, diluted with water to a fixed volume.
[0008] Specifically, the effective viable count of *Bacillus cereus* bacterial solution in the compound microbial agent is ≥2.0 × 10⁻⁶. 9 CFU / mL, and the number of viable bacteria in *Priscilla megaterium* culture ≥1.0 × 10⁻⁶. 8 CFU / mL, effective viable count of Bacillus saprophyticus in bacterial culture ≥1.5×10⁻⁶ 9 cfu / mL.
[0009] This invention provides a method for preparing the above-mentioned composite microbial agent, which mainly includes the following steps: Step 1, Strain activation and seed culture: Bacillus porchuanensis (purchased from China General Microbiological Culture Collection Center, strain number CGMCC1.8803) was activated according to the instructions and inoculated into a special culture medium (10.0g peptone, 3.0g beef extract, 5.0g NaCl, 15.0g agar, 1.0L distilled water, pH 7.0-7.2) and cultured in a shaker at 30℃ for 24-30h. Then, it was transferred to expansion culture medium I (10.0g peptone, 10.0g yeast extract, 15.0g glucose, 1.5g KH2PO4, 2.0g (NH4)2SO4, 0.5g MgSO4·7H2O, 2.0g NaCl, 1.0g L-glutamate, 1L distilled water, pH 7.0-7.2) for expansion culture to obtain Bacillus porchuanensis seed culture with an OD value of 0.6-0.8. After activation according to the instructions, *Priestella megaterium* (purchased from China General Microbiological Culture Collection Center, strain number CGMCC1.8802) was inoculated into a special culture medium (10.0 g peptone, 3.0 g beef extract, 5.0 g NaCl, 15.0 g agar, 1.0 L distilled water, pH 7.0-7.2) and cultured in a shaker at 30°C for 24-30 h. Then, at an inoculation rate of 8-10% (v / v), it was transferred to expansion culture medium II (15.0 g glucose, 5 g glycerol, 3.0 g beef extract, 2.0 g (NH4)2SO4, 1 L distilled water, pH 7.0-7.2) for expansion culture to obtain *Priestella megaterium* seed culture with an OD value of 0.8-1.0. Bacillus saprophyticus (purchased from China General Microbiological Culture Collection Center, strain number CGMCC1.9055) was activated according to the instructions and then inoculated into a special culture medium (10.0g peptone, 3.0g beef extract, 5.0g NaCl, 15.0g agar, 1.0L distilled water, pH 7.0-7.2). The medium was incubated at 30℃ in a shaker for 24-30h. Then, at a 5-8% (v / v) inoculation rate, the medium was transferred to expansion medium III (15g peptone, 5.0g yeast extract, 10.0g glucose, 1.5g K₂HPO₄, 0.5g KH₂PO₄, 1L distilled water, pH 7.0-7.2) for expansion culture to obtain a seed culture of Bacillus saprophyticus with an OD value of 0.6-0.8. Step 2, Fermentation and Propagation: Inoculate the *Bacillus thuringiensis* seed culture at a rate of 5-8% (v / v) into fermentation medium I (soluble starch 20-40g, soybean meal powder 10-20g, yeast extract 5.0g, K₂HPO₄·3H₂O 4.0g, MgSO₄·7H₂O 1.0g, KH₂PO₄ 1.5g, CaCO₃ 3.0-5.0g, distilled water 1L, pH 7.0-7.2). Control the fermentation temperature at approximately 30℃, aeration rate of 1:1 (v / v min), and stirring speed of 180-220 rpm. Fermentation should continue until the effective viable cell count is ≥2.2 × 10⁻⁶. 9 CFU / mL, centrifugation yielded an effective viable count ≥2.0 × 10⁻⁶. 9 CFU / mL of Bacillus hygroscopicus culture; Inoculate *Priscilla megaterium* seed culture at an inoculum rate of 8-10% (v / v) into fermentation medium II (40-60g glucose, 15g peptone, 6.0g K₂HPO₄, 3.0g KH₂PO₄, 0.1g CaCl₂·2H₂O, 0.01g FeSO₄·7H₂O, 1L distilled water, pH 7.0-7.2). Control the fermentation temperature at approximately 30℃, aeration rate of 1:1.5 (v / v min), and stirring speed of 150-180 rpm. Fermentation should continue until the effective viable cell count is ≥1.2 × 10⁻⁶. 8 CFU / mL, centrifugation yielded an effective viable count ≥1.0 × 10⁻⁶. 8 CFU / mL Priestella gianti bacterial suspension; Inoculate the Bacillus saprophyticus seed culture at a rate of 5-8% (v / v) into fermentation medium III (25.0g soybean meal, 5.0g wheat bran, 2.0g (NH4)2SO4, 1.0g KH2PO4, 0.3g MgSO4·7H2O, 0.5g CaCO3, 1L distilled water, pH 7.0-7.2). Control the fermentation temperature at approximately 30℃, the aeration rate at 1:1.2 (v / v min), and the stirring speed at 200-220 rpm. Fermentation should continue until the effective viable count reaches ≥1.8 × 10⁻⁶. 9 CFU / mL, centrifugation yielded an effective viable count ≥1.5 × 10⁻⁶. 9 CFU / mL of Bacillus rotundus bacterial solution; Step 3, prepare compound microbial inoculant: Take 50-60 mL of the above-prepared *Bacillus puerariae* bacterial suspension, 80-100 mL of *Priscilla megaterium* bacterial suspension, 50-60 mL of *Bacillus humifusa* bacterial suspension, 10-15 g of resistant dextrin, 80-100 g of glycerol, 20-30 g of trehalose, and 8-10 g of sodium ascorbate. Add an appropriate amount of water and stir to dissolve. Then add 20-30 g of *Tremella fuciformis* oligopeptide, 40-50 g of *Sanghuang* polysaccharide, 10-15 mL of sugar alcohol silicon, 15-20 mL of sugar alcohol zinc, and 20-30 g of sodium polyglutamate. Dissolve in water and bring the volume to 1 L.
[0010] The present invention provides a specific method of using the above-mentioned compound microbial agent: it can be applied by root irrigation or foliar spraying.
[0011] As a preferred method, the above-mentioned compound microbial agent is diluted 50-60 times and applied to the roots during the honeysuckle germination period.
[0012] As a preferred method, the above-mentioned compound microbial agent is diluted 100-120 times and then sprayed on the leaves at the early stage of honeysuckle bud formation.
[0013] The present invention has the following beneficial effects: The compound microbial agent prepared in this invention contains *Bacillus buergerianum* and *Bacillus saprophyticus*, which can cooperate with *Priscilla megaterium* to rapidly colonize the roots of honeysuckle, seize ecological niches, and directly inhibit the infection of soil-borne pathogens by competing for nutrients and space. Simultaneously, they can produce antibacterial substances such as ester peptides, directly inhibiting or killing pathogens. *Tremella fuciformis* oligopeptides and *Phellinus linteus* polysaccharides are fungal extracts that, while inhibiting pathogens, can induce honeysuckle to produce defenses, enhancing its defensive capabilities. Silicon, zinc, and other components can repair honeysuckle wounds and prevent further infection by pathogens. Resistant dextrin, glycerol, trehalose, and ascorbic acid constitute a biochemical barrier, protecting microorganisms while ensuring the health of honeysuckle cells, maintaining normal cellular physiological functions, removing harmful substances, and reducing disease damage. Sodium polyglutamate can form a protective film on the roots and leaves of honeysuckle, delaying the persistence of the agent.
[0014] The composite microbial agent of this invention utilizes the synergistic effect of microorganisms and various components to comprehensively enhance the disease resistance of honeysuckle from both internal and external perspectives, mobilize and strengthen the honeysuckle's own defense capabilities, and achieve a lasting control effect against common honeysuckle diseases. Detailed Implementation
[0015] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. After reading the present invention, any modifications of the present invention in various equivalent forms by those skilled in the art will fall within the scope of protection of the claims of this application.
[0016] Example 1: Preparation of compound microbial inoculant (1L) Step 1, Strain activation and seed culture: Bacillus porgyensis (purchased from China General Microbiological Culture Collection Center, strain number CGMCC1.8803) was activated according to the instructions and inoculated into a special culture medium (10.0 g peptone, 3.0 g beef extract, 5.0 g NaCl, 15.0 g agar, 1.0 L distilled water, pH 7.0) and cultured in a shaker at 30°C for 30 h. Then, it was transferred to expansion culture medium I (10.0 g peptone, 10.0 g yeast extract, 15.0 g glucose, 1.5 g KH2PO4, 2.0 g (NH4)2SO4, 0.5 g MgSO4·7H2O, 2.0 g NaCl, 1.0 g L-glutamate, 1 L distilled water, pH 7.0) at a 5% (v / v) inoculation rate for expansion culture to obtain Bacillus porgyensis seed culture with an OD value of 0.6. After activation according to the instructions, *Priestella megaterium* (purchased from China General Microbiological Culture Collection Center, strain number CGMCC1.8802) was inoculated into a special culture medium (10.0 g peptone, 3.0 g beef extract, 5.0 g NaCl, 15.0 g agar, 1.0 L distilled water, pH 7.0) and cultured in a shaker at 30°C for 30 h. Then, at an inoculation rate of 8% (v / v), it was transferred to expansion medium II (15.0 g glucose, 5 g glycerol, 3.0 g beef extract, 2.0 g (NH4)2SO4, 1 L distilled water, pH 7.0) for expansion culture to obtain *Priestella megaterium* seed culture with an OD value of 1.0. Bacillus rotundus (purchased from China General Microbiological Culture Collection Center, strain number CGMCC1.9055) was activated according to the instructions and then inoculated into a special culture medium (10.0 g peptone, 3.0 g beef extract, 5.0 g NaCl, 15.0 g agar, 1.0 L distilled water, pH 7.0). It was cultured in a shaker at 30°C for 24 h, and then transferred to expansion medium III (15 g peptone, 5.0 g yeast extract, 10.0 g glucose, 1.5 g K2HPO4, 0.5 g KH2PO4, 1 L distilled water, pH 7.0) at an inoculation rate of 8% (v / v) for expansion culture to obtain Bacillus rotundus seed culture with an OD value of 0.6. Step 2, Fermentation and Propagation: The *Bacillus thuringiensis* seed culture from Baochuan was inoculated at an 8% (v / v) in fermentation medium I (40g soluble starch, 10g soybean meal, 5.0g yeast extract, 4.0g K₂HPO₄·3H₂O, 1.0g MgSO₄·7H₂O, 1.5g KH₂PO₄, 5.0g CaCO₃, 1L distilled water, pH 7.0). The fermentation temperature was controlled at approximately 30℃, the aeration rate was 1:1 (v / v min), and the stirring speed was 220 rpm. Fermentation was carried out until the effective viable cell count was ≥2.2 × 10⁻⁶. 9 CFU / mL, centrifugation yielded an effective viable count ≥2.0 × 10⁻⁶. 9 CFU / mL of Bacillus hygroscopicus culture; The *Priestella megaterium* seed culture was inoculated at an 8% (v / v) incubation rate into fermentation medium II (60 g glucose, 15 g peptone, 6.0 g K₂HPO₄, 3.0 g KH₂PO₄, 0.1 g CaCl₂·2H₂O, 0.01 g FeSO₄·7H₂O, 1 L distilled water, pH 7.0). The fermentation temperature was controlled at approximately 30°C, the aeration rate was 1:1.5 (v / v min), and the stirring speed was 150 rpm. Fermentation was carried out until the effective viable cell count was ≥1.2 × 10⁻⁶. 8 CFU / mL, centrifugation yielded an effective viable count ≥1.0 × 10⁻⁶. 8CFU / mL Priestella gianti bacterial suspension; The Bacillus saprophyticus seed culture was inoculated at an 8% (v / v) in fermentation medium III (25.0 g soybean meal, 5.0 g wheat bran, 2.0 g (NH4)2SO4, 1.0 g KH2PO4, 0.3 g MgSO4·7H2O, 0.5 g CaCO3, 1 L distilled water, pH 7.0). The fermentation temperature was controlled at approximately 30℃, the aeration rate was 1:1.2 (v / v min), and the stirring speed was 220 rpm. Fermentation was carried out until the effective viable count was ≥1.8 × 10⁻⁶. 9 CFU / mL, centrifugation yielded an effective viable count ≥1.5 × 10⁻⁶. 9 CFU / mL of Bacillus rotundus bacterial solution; Step 3, prepare compound microbial inoculant: Take 50 mL of the above-prepared *Bacillus puerariae* bacterial solution, 80 mL of *Priscilla megaterium* bacterial solution, 50 mL of *Bacillus humifusa* bacterial solution, 10 g of resistant dextrin, 80 g of glycerol, 20 g of trehalose, and 8 g of sodium ascorbate, add an appropriate amount of water and stir to dissolve. Then add 20 g of *Tremella fuciformis* oligopeptide, 40 g of *Sanghuang* polysaccharide, 10 mL of sugar alcohol silicon, 15 mL of sugar alcohol zinc, and 20 g of sodium polyglutamate. Dissolve in water and bring the volume to 1 L to obtain the compound microbial agent.
[0017] Example 2 Preparation of compound microbial inoculant (1L) Step 1, Strain activation and seed culture: Bacillus porgyensis (purchased from China General Microbiological Culture Collection Center, strain number CGMCC1.8803) was activated according to the instructions and inoculated into a special culture medium (10.0 g peptone, 3.0 g beef extract, 5.0 g NaCl, 15.0 g agar, 1.0 L distilled water, pH 7.2) and cultured in a shaker at 30°C for 24 h. Then, it was transferred to expansion culture medium I (10.0 g peptone, 10.0 g yeast extract, 15.0 g glucose, 1.5 g KH2PO4, 2.0 g (NH4)2SO4, 0.5 g MgSO4·7H2O, 2.0 g NaCl, 1.0 g L-glutamate, 1 L distilled water, pH 7.2) at an inoculation rate of 8% (v / v) for expansion culture to obtain Bacillus porgyensis seed culture with an OD value of 0.8. After activation according to the instructions, *Priestella megaterium* (purchased from China General Microbiological Culture Collection Center, strain number CGMCC1.8802) was inoculated into a special culture medium (10.0 g peptone, 3.0 g beef extract, 5.0 g NaCl, 15.0 g agar, 1.0 L distilled water, pH 7.2) and cultured in a shaker at 30°C for 24 h. Then, at a 10% (v / v) inoculation rate, it was transferred to expansion medium II (15.0 g glucose, 5 g glycerol, 3.0 g beef extract, 2.0 g (NH4)2SO4, 1 L distilled water, pH 7.2) for expansion culture to obtain *Priestella megaterium* seed culture with an OD value of 0.8. Bacillus rotundus (purchased from China General Microbiological Culture Collection Center, strain number CGMCC1.9055) was activated according to the instructions and inoculated into a special culture medium (10.0 g peptone, 3.0 g beef extract, 5.0 g NaCl, 15.0 g agar, 1.0 L distilled water, pH 7.2). It was cultured in a shaker at 30°C for 30 h. Then, it was transferred to expansion culture medium III (15 g peptone, 5.0 g yeast extract, 10.0 g glucose, 1.5 g K2HPO4, 0.5 g KH2PO4, 1 L distilled water, pH 7.2) at a 5% (v / v) inoculation rate for expansion culture to obtain Bacillus rotundus seed culture with an OD value of 0.8. Step 2, Fermentation and Propagation: The *Bacillus thuringiensis* seed culture was inoculated at a rate of 5% (v / v) into fermentation medium I (20g soluble starch, 20g soybean meal, 5.0g yeast extract, 4.0g K₂HPO₄·3H₂O, 1.0g MgSO₄·7H₂O, 1.5g KH₂PO₄, 3.0g CaCO₃, 1L distilled water, pH 7.2). The fermentation temperature was controlled at approximately 30℃, the aeration rate was 1:1 (v / v min), and the stirring speed was 180 rpm. Fermentation was carried out until the effective viable cell count was ≥2.2 × 10⁻⁶. 9 CFU / mL, centrifugation yielded an effective viable count ≥2.0 × 10⁻⁶. 9 CFU / mL of Bacillus hygroscopicus culture; The *Priestella megaterium* seed culture was inoculated at a rate of 10% (v / v) into fermentation medium II (40 g glucose, 15 g peptone, 6.0 g K₂HPO₄, 3.0 g KH₂PO₄, 0.1 g CaCl₂·2H₂O, 0.01 g FeSO₄·7H₂O, 1 L distilled water, pH 7.2). The fermentation temperature was controlled at approximately 30°C, the aeration rate was 1:1.5 (v / v min), and the stirring speed was 180 rpm. Fermentation was carried out until the effective viable cell count was ≥1.2 × 10⁻⁶. 8 CFU / mL, centrifugation yielded an effective viable count ≥1.0 × 10⁻⁶. 8CFU / mL Priestella gianti bacterial suspension; The Bacillus saprophyticus seed culture was inoculated at a rate of 5% (v / v) into fermentation medium III (25.0 g soybean meal, 5.0 g wheat bran, 2.0 g (NH4)2SO4, 1.0 g KH2PO4, 0.3 g MgSO4·7H2O, 0.5 g CaCO3, 1 L distilled water, pH 7.2). The fermentation temperature was controlled at approximately 30℃, the aeration rate was 1:1.2 (v / v min), and the stirring speed was 200 rpm. Fermentation was carried out until the effective viable count was ≥1.8 × 10⁻⁶. 9 CFU / mL, centrifugation yielded an effective viable count ≥1.5 × 10⁻⁶. 9 CFU / mL of Bacillus rotundus bacterial solution; Step 3, prepare compound microbial inoculant: Take 60 mL of the above-prepared *Bacillus puerariae* bacterial solution, 100 mL of *Priscilla megaterium* bacterial solution, 60 mL of *Bacillus humifusa* bacterial solution, 15 g of resistant dextrin, 100 g of glycerol, 30 g of trehalose, and 10 g of sodium ascorbate, add an appropriate amount of water and stir to dissolve. Then add 30 g of *Tremella fuciformis* oligopeptide, 50 g of *Sanghuang* polysaccharide, 15 mL of sugar alcohol silicon, 20 mL of sugar alcohol zinc, and 30 g of sodium polyglutamate. Dissolve in water and bring the volume to 1 L to obtain the compound microbial agent.
[0018] Example 3 Preparation of compound microbial inoculant (1L) Step 1, Strain activation and seed culture: Bacillus porgyensis (purchased from China General Microbiological Culture Collection Center, strain number CGMCC1.8803) was activated according to the instructions and inoculated into a special culture medium (10.0 g peptone, 3.0 g beef extract, 5.0 g NaCl, 15.0 g agar, 1.0 L distilled water, pH 7.0) and cultured in a shaker at 30°C for 30 h. Then, it was transferred to expansion culture medium I (10.0 g peptone, 10.0 g yeast extract, 15.0 g glucose, 1.5 g KH2PO4, 2.0 g (NH4)2SO4, 0.5 g MgSO4·7H2O, 2.0 g NaCl, 1.0 g L-glutamate, 1 L distilled water, pH 7.0) at a 6% (v / v) inoculation rate for expansion culture to obtain Bacillus porgyensis seed culture with an OD value of 0.8. After activation according to the instructions, *Priestella megaterium* (purchased from China General Microbiological Culture Collection Center, strain number CGMCC1.8802) was inoculated into a special culture medium (10.0 g peptone, 3.0 g beef extract, 5.0 g NaCl, 15.0 g agar, 1.0 L distilled water, pH 7.0) and cultured in a shaker at 30°C for 30 h. Then, at an inoculation rate of 8% (v / v), it was transferred to expansion medium II (15.0 g glucose, 5 g glycerol, 3.0 g beef extract, 2.0 g (NH4)2SO4, 1 L distilled water, pH 7.0) for expansion culture to obtain *Priestella megaterium* seed culture with an OD value of 0.8. Bacillus rotundus (purchased from China General Microbiological Culture Collection Center, strain number CGMCC1.9055) was activated according to the instructions and inoculated into a special culture medium (10.0 g peptone, 3.0 g beef extract, 5.0 g NaCl, 15.0 g agar, 1.0 L distilled water, pH 7.0) and cultured in a shaker at 30°C for 24 h. Then, it was transferred to expansion culture medium III (15 g peptone, 5.0 g yeast extract, 10.0 g glucose, 1.5 g K2HPO4, 0.5 g KH2PO4, 1 L distilled water, pH 7.0) at an inoculation rate of 8% (v / v) for expansion culture to obtain Bacillus rotundus seed culture with an OD value of 0.8. Step 2, Fermentation and Propagation: The *Bacillus hygroscopicus* seed culture from Baochuan was inoculated at a rate of 6% (v / v) into fermentation medium I (30g soluble starch, 15g soybean meal, 5.0g yeast extract, 4.0g K₂HPO₄·3H₂O, 1.0g MgSO₄·7H₂O, 1.5g KH₂PO₄, 4.0g CaCO₃, 1L distilled water, pH 7.0). The fermentation temperature was controlled at approximately 30℃, the aeration rate was 1:1 (v / v min), and the stirring speed was 200 rpm. Fermentation was carried out until the effective viable cell count was ≥2.2 × 10⁻⁶. 9 CFU / mL, centrifugation yielded an effective viable count ≥2.0 × 10⁻⁶. 9 CFU / mL of Bacillus hygroscopicus culture; The Priestella megaterium seed culture was inoculated at a rate of 9% (v / v) into fermentation medium II (50g glucose, 15g peptone, 6.0g K₂HPO₄, 3.0g KH₂PO₄, 0.1g CaCl₂·2H₂O, 0.01g FeSO₄·7H₂O, 1L distilled water, pH 7.0). The fermentation temperature was controlled at approximately 30℃, the aeration rate was 1:1.5 (v / v min), and the stirring speed was 170 rpm. Fermentation was carried out until the effective viable cell count was ≥1.2 × 10⁻⁶. 8 CFU / mL, centrifugation yielded an effective viable count ≥1.0 × 10⁻⁶. 8CFU / mL Priestella gianti bacterial suspension; The Bacillus saprophyticus seed culture was inoculated at a rate of 7% (v / v) into fermentation medium III (25.0 g soybean meal, 5.0 g wheat bran, 2.0 g (NH4)2SO4, 1.0 g KH2PO4, 0.3 g MgSO4·7H2O, 0.5 g CaCO3, 1 L distilled water, pH 7.0). The fermentation temperature was controlled at approximately 30℃, the aeration rate was 1:1.2 (v / v min), and the stirring speed was 210 rpm. Fermentation was carried out until the effective viable count was ≥1.8 × 10⁻⁶. 9 CFU / mL, centrifugation yielded an effective viable count ≥1.5 × 10⁻⁶. 9 CFU / mL of Bacillus rotundus bacterial solution; Step 3, prepare compound microbial inoculant: Take 55 mL of the above-prepared *Bacillus puerariae* bacterial solution, 90 mL of *Priscilla megaterium* bacterial solution, 55 mL of *Bacillus humifusa* bacterial solution, 13 g of resistant dextrin, 90 g of glycerol, 25 g of trehalose, and 9 g of sodium ascorbate, add an appropriate amount of water and stir to dissolve. Then add 25 g of *Tremella fuciformis* oligopeptide, 45 g of *Sanghuang* polysaccharide, 13 mL of sugar alcohol silicon, 18 mL of sugar alcohol zinc, and 25 g of sodium polyglutamate. Dissolve in water and bring the volume to 1 L to obtain the compound microbial agent.
[0019] Comparative Example 1: Preparation of Compound Microbial Agent (1L) Step 1, Strain activation and seed culture: After activation according to the instructions, *Priestella megaterium* (purchased from China General Microbiological Culture Collection Center, strain number CGMCC1.8802) was inoculated into a special culture medium (10.0 g peptone, 3.0 g beef extract, 5.0 g NaCl, 15.0 g agar, 1.0 L distilled water, pH 7.0) and cultured in a shaker at 30°C for 30 h. Then, at an inoculation rate of 8% (v / v), it was transferred to an expansion culture medium (15.0 g glucose, 5 g glycerol, 3.0 g beef extract, 2.0 g (NH4)2SO4, 1 L distilled water, pH 7.0) for expansion culture to obtain *Priestella megaterium* seed culture with an OD value of 1.0. Step 2, Fermentation and Propagation: The Priestella megaterium seed culture was inoculated into the fermentation medium (60 g glucose, 15 g peptone, 6.0 g K₂HPO₄, 3.0 g KH₂PO₄, 0.1 g CaCl₂·2H₂O, 0.01 g FeSO₄·7H₂O, 1 L distilled water, pH 7.0) at an 8% (v / v) inoculation rate. The fermentation temperature was controlled at approximately 30°C, the aeration rate was 1:1.5 (v / v min), and the stirring speed was 150 rpm. Fermentation was carried out until the effective viable cell count was ≥1.2 × 10⁻⁶. 8CFU / mL, centrifugation yielded an effective viable count ≥1.0 × 10⁻⁶. 8 CFU / mL Priestella gianti bacterial suspension; Step 3, prepare compound microbial inoculant: Take 80 mL of the prepared Priestella giantiflora bacterial solution, 10 g of resistant dextrin, 80 g of glycerol, 20 g of trehalose, and 8 g of sodium ascorbate, add an appropriate amount of water and stir to dissolve. Then add 20 g of Tremella fuciformis oligopeptide, 40 g of Phellinus linteus polysaccharide, 10 mL of sugar alcohol silicon, 15 mL of sugar alcohol zinc, and 20 g of sodium polyglutamate. Dissolve in water and bring the volume to 1 L to obtain the compound microbial agent.
[0020] Comparative Example 2: Preparation of Compound Microbial Agent (1L) Step 1, Strain activation and seed culture: Bacillus porgyensis (purchased from China General Microbiological Culture Collection Center, strain number CGMCC1.8803) was activated according to the instructions and inoculated into a special culture medium (10.0 g peptone, 3.0 g beef extract, 5.0 g NaCl, 15.0 g agar, 1.0 L distilled water, pH 7.0) and cultured in a shaker at 30°C for 30 h. Then, it was transferred to expansion culture medium I (10.0 g peptone, 10.0 g yeast extract, 15.0 g glucose, 1.5 g KH2PO4, 2.0 g (NH4)2SO4, 0.5 g MgSO4·7H2O, 2.0 g NaCl, 1.0 g L-glutamate, 1 L distilled water, pH 7.0) at a 5% (v / v) inoculation rate for expansion culture to obtain Bacillus porgyensis seed culture with an OD value of 0.6. After activation according to the instructions, *Priestella megaterium* (purchased from China General Microbiological Culture Collection Center, strain number CGMCC1.8802) was inoculated into a special culture medium (10.0 g peptone, 3.0 g beef extract, 5.0 g NaCl, 15.0 g agar, 1.0 L distilled water, pH 7.0) and cultured in a shaker at 30°C for 30 h. Then, at an inoculation rate of 8% (v / v), it was transferred to expansion medium II (15.0 g glucose, 5 g glycerol, 3.0 g beef extract, 2.0 g (NH4)2SO4, 1 L distilled water, pH 7.0) for expansion culture to obtain *Priestella megaterium* seed culture with an OD value of 1.0. Step 2, Fermentation and Propagation: The *Bacillus thuringiensis* seed culture from Baochuan was inoculated at an 8% (v / v) in fermentation medium I (40g soluble starch, 10g soybean meal, 5.0g yeast extract, 4.0g K₂HPO₄·3H₂O, 1.0g MgSO₄·7H₂O, 1.5g KH₂PO₄, 5.0g CaCO₃, 1L distilled water, pH 7.0). The fermentation temperature was controlled at approximately 30℃, the aeration rate was 1:1 (v / v min), and the stirring speed was 220 rpm. Fermentation was carried out until the effective viable cell count was ≥2.2 × 10⁻⁶. 9 CFU / mL, centrifugation yielded an effective viable count ≥2.0 × 10⁻⁶. 9 CFU / mL of Bacillus hygroscopicus culture; The *Priestella megaterium* seed culture was inoculated at an 8% (v / v) incubation rate into fermentation medium II (60 g glucose, 15 g peptone, 6.0 g K₂HPO₄, 3.0 g KH₂PO₄, 0.1 g CaCl₂·2H₂O, 0.01 g FeSO₄·7H₂O, 1 L distilled water, pH 7.0). The fermentation temperature was controlled at approximately 30°C, the aeration rate was 1:1.5 (v / v min), and the stirring speed was 150 rpm. Fermentation was carried out until the effective viable cell count was ≥1.2 × 10⁻⁶. 8 CFU / mL, centrifugation yielded an effective viable count ≥1.0 × 10⁻⁶. 8 CFU / mL Priestella gianti bacterial suspension; Step 3, prepare compound microbial inoculant: Take 50 mL of the prepared *Bacillus buergerianus* bacterial solution, 80 mL of *Priscilla megaterium* bacterial solution, 10 g of resistant dextrin, 80 g of glycerol, 20 g of trehalose, and 8 g of sodium ascorbate, add an appropriate amount of water and stir to dissolve. Then add 10 mL of sugar alcohol silicon, 15 mL of sugar alcohol zinc, and 20 g of sodium polyglutamate, dissolve in water, and bring the volume to 1 L to obtain the compound microbial agent.
[0021] Performance testing I. The role of compound microbial agents in improving the disease resistance of honeysuckle 1. Experiment and Methods The honeysuckle planted in this experiment was a four-year-old honeysuckle plantation in Pingyi County, Linyi City.
[0022] Five experimental zones were set up, each containing 30 honeysuckle plants. The plants were treated as follows: Treatment Group 1: The compound microbial inoculant obtained in Example 1 was applied to the roots during the germination period (diluted 60 times), and the rest was handled with conventional field management. Treatment group 2: The compound microbial agent obtained in Example 1 was applied by root irrigation during the germination period (diluted 60 times) and foliar spraying at the early bud stage (diluted 120 times), with the rest being conventional field management; Treatment group 3: The compound microbial agent obtained from Comparative Example 1 was applied by root irrigation during the germination period (diluted 60 times) and foliar spraying at the early bud stage (diluted 120 times), and the rest were conventional field management; Treatment group 4: The compound microbial agent obtained from Comparative Example 2 was applied by root irrigation during the germination period (diluted 60 times) and foliar spraying at the early bud stage (diluted 120 times), and the rest were treated with conventional field management; Control group: Routine field management.
[0023] The incidence and control effects of root rot, brown spot, and powdery mildew in honeysuckle were counted two months after root irrigation and one month after foliar spraying.
[0024] 2. Results and Analysis As shown in Tables 1 and 2, the compound microbial agent prepared in this invention has good control effects on honeysuckle root rot, brown spot disease, and powdery mildew, significantly reducing their incidence. In treatment group 1, only root irrigation during the germination period is used. Treatment groups 3 and 4 are different from the compound microbial agent of this invention, and their control effects are relatively poor. Furthermore, the types of microorganisms in treatment groups 3 and 4 are different, and their control effects on different diseases are significantly different.
[0025] Table 1 Comparison of disease incidence in honeysuckle under different treatments Table 2 Comparison of control effects of different treatments on honeysuckle
Claims
1. A compound microbial inoculant for improving the disease resistance of honeysuckle, characterized in that, 1L of compound microbial agent contains the following components: 50-60mL of Bacillus puerariae bacterial solution, 80-100mL of Priestella megaterium bacterial solution, 50-60mL of Bacillus humifusa bacterial solution, 10-15g of resistant dextrin, 80-100g of glycerol, 20-30g of trehalose, 8-10g of sodium ascorbate, 20-30g of Tremella fuciformis oligopeptide, 40-50g of Phellinus linteus polysaccharide, 10-15mL of sugar alcohol silicon, 15-20mL of sugar alcohol zinc, and 20-30g of sodium polyglutamate, diluted with water to volume.
2. The compound microbial agent as described in claim 1, characterized in that, The effective viable count of *Bacillus puerariae* bacterial solution in the compound microbial agent is ≥2.0 × 10⁻⁶. 9 CFU / mL, and the number of viable bacteria in *Priscilla megaterium* culture ≥1.0 × 10⁻⁶. 8 CFU / mL, effective viable count of Bacillus saprophyticus in bacterial culture ≥1.5×10⁻⁶ 9 cfu / mL.
3. The compound microbial agent as described in claim 1, characterized in that, The preparation method of the compound microbial agent includes the following steps: Step 1: After activating the purchased CGMCC 1.8803 Bacillus thuringiensis, it was inoculated into a special culture medium and cultured in a shaker at 30°C for 24-30 hours. Then, it was transferred to expansion culture medium I at an inoculation rate of 5-8% (v / v) to obtain Bacillus thuringiensis seed culture. After activating the purchased CGMCC 1.8802 Priestella megaterium, it was inoculated into a special culture medium and cultured in a shaker at 30°C for 24-30 hours. Then, it was transferred to expansion culture medium II at an inoculation rate of 8-10% (v / v) to obtain Priestella megaterium seed culture. After activating the purchased CGMCC 1.9055 Bacillus rotundus, it was inoculated into a special culture medium and cultured in a shaker at 30°C for 24-30 hours. Then, it was transferred to expansion culture medium III at an inoculation rate of 5-8% (v / v) to obtain Bacillus rotundus seed culture. Step 2: The *Bacillus hygroscopicus* seed culture was inoculated into fermentation medium I at an inoculation rate of 5-8% (v / v) to obtain an effective viable count ≥2.0 × 10⁻⁶. 9 CFU / mL of Bacillus hygroscopicus culture; The Priestella megaterium seed culture was inoculated into fermentation medium II at an inoculum rate of 8-10% (v / v) to obtain an effective viable count ≥1.0 × 10⁻⁶. 8 CFU / mL Priestella gianti bacterial suspension; The Bacillus saprophyticus seed culture was inoculated into fermentation medium III at an inoculation rate of 5-8% (v / v) to obtain an effective viable count ≥1.5×10⁻⁶. 9 CFU / mL of Bacillus rotundus bacterial solution; Step 3: Take 50-60 mL of *Bacillus puerariae* bacterial suspension, 80-100 mL of *Priscilla megaterium* bacterial suspension, 50-60 mL of *Bacillus humifusa* bacterial suspension, 10-15 g of resistant dextrin, 80-100 g of glycerol, 20-30 g of trehalose, and 8-10 g of sodium ascorbate, dissolve them in water, then add 20-30 g of *Tremella fuciformis* oligopeptide, 40-50 g of *Sanghuang* polysaccharide, 10-15 mL of sugar alcohol silicon, 15-20 mL of sugar alcohol zinc, and 20-30 g of sodium polyglutamate, dissolve in water and bring the volume to 1 L to obtain the compound microbial agent.
4. The compound microbial agent as described in claim 3, characterized in that, In step 1 of the preparation method, the OD values of the Bacillus hygroscopicus seed liquid are 0.6~0.8, the OD values of the Priestella megaterium seed liquid are 0.8~1.0, and the OD values of the Bacillus humifusa seed liquid are 0.6~0.
8.
5. The compound microbial agent as described in claim 1, characterized in that, The compound microbial agent is applied by root irrigation and / or foliar spraying.
6. The compound microbial agent as described in claim 5, characterized in that, The compound microbial agent was diluted 50-60 times and applied to the roots during the honeysuckle germination period.
7. The composite microbial agent as described in claim 5, characterized in that, The compound microbial agent was diluted 100-120 times and sprayed on the leaves of honeysuckle in the early stage of flower bud formation.